Project Grant R43HG014432

DEVELOPMENT OF CHROMEX30 FOR NANOSCALE IMAGING OF GENETIC ORGANIZATION AND DISEASE PHENOTYPES - PROJECT SUMMARY ASSESSING THE STRUCTURE OF GENETIC MATERIAL AND GENETIC DISORDERS AT THE NANOSCALE (~10 NM) AND EVALUATING THE 3D DISTRIBUTION OF MOLECULAR BIOMARKERS IN THAT CONTEXT IS CURRENTLY SEVERELY LIMITED AS IT DEPENDS ON EXPENSIVE, TIME-CONSUMING AND EXTREMELY COMPLEX CORRELATIVE LIGHT AND ELECTRON MICROSCOPY (CLEM) AS THE ONLY SUITABLE TOOL. PANLUMINATE INC. HAS DEVELOPED A PROPRIETARY NEW CELL AND TISSUE EXPANSION TECHNOLOGY WE CALL PAN-EXPANSION MICROSCOPY (PAN-EXM), THAT DIRECTLY ADDRESSES THIS CHALLENGE. BY COMBINING ~16-FOLD LINEAR EXPANSION OF BIOLOGICAL SAMPLES WITH NOVEL BULK (PAN) STAINING OF AMINO ACIDS, PAN-EXM ENABLES THE MAPPING OF THE 3D NANOSCALE DISTRIBUTION OF SPECIFIC BIOMARKERS WITHIN THE ULTRASTRUCTURE OF CELLS. HALLMARK ULTRASTRUCTURAL FEATURES, SUCH AS CHROMATIN FIBERS AND NUCLEAR ENVELOPES, CAN NOW BE RESOLVED BY THEIR CHARACTERISTIC PAN-STAINING PATTERN, ANALOGOUS TO HEAVY-METAL STAINS IN ELECTRON MICROSCOPY (EM). IN CONTRAST TO EM/CLEM, HOWEVER, PAN-EXM IS AN ALL-OPTICAL METHOD THAT ALLOWS RESEARCHERS TO INTERROGATE BIOLOGICAL SAMPLES USING STANDARD CONFOCAL MICROSCOPES; THIS OFFERS IMPORTANT ADVANTAGES IN TERMS OF TIME, COST, COMPLEXITY AND SAMPLE PRESERVATION VERSUS EM ALTERNATIVES. OUR PROPOSED PHASE I PROJECT IS TO DEVELOP CHROMEX30, A NOVEL VARIANT OF PAN-EXM SPECIFICALLY TAILORED FOR GENOMICS RESEARCH THAT OFFERS TWICE THE RESOLUTION. IN PARTICULAR, WE WILL: (1) DEVELOP, OPTIMIZE, AND VALIDATE CHROMEX30, A >30X EXPANSION PROTOCOL FOR CHARACTERIZING THE ULTRASTRUCTURE OF GENETIC ORGANIZATION. WE WILL INTRODUCE A NOVEL POLYMER SYSTEM THAT ACHIEVES RAPID >30X EXPANSION FOR SUB-10 NM SPATIAL RESOLUTION IMAGING WHILE RETAINING PROTEINS AND MAINTAINING ISOTROPIC 3D EXPANSION; AND (2) VALIDATE CHROMEX30 AS AN ULTRASTRUCTURAL PHENOTYPING TOOL AT THE EXAMPLE OF GREENBERG DYSPLASIA USING CONTROL AND LAMIN B RECEPTOR (LBR) KNOCKDOWN HL-60/S4 CELLS. THIS WORK WILL VALIDATE CHROMEX30 AS A KEY TOOL FOR STUDYING GENETIC DISEASE PHENOTYPES AND LAY THE FOUNDATION FOR A PHASE II PROJECT TO ESTABLISH IT AS A LEADING IMAGING TECHNOLOGY IN GENETICS. OUR LONG-TERM GOAL IS FOR CHROMEX30 TO BECOME A CORE TECHNOLOGY FOR CREATING COMPREHENSIVE GENOMIC ATLASES AND A PREFERRED METHOD FOR TARGETED RESEARCH AND DIAGNOSTICS, ADVANCING PRECISION MEDICINE ACROSS VARIOUS FIELDS.